启动子CMV和EF1α对人胰岛素基因在BHK细胞中表达的影响

The Difference between the Promoter CMV and EF1αto the Expression of Human Insulin Gene in BHK Cell

目的 构建人胰岛素基因真核高效表达载体 ,为胰岛素转基因研究奠定基础。方法 用限制性内切酶SpeⅠ和HindⅢ消化pEF1α GFP ,回收 1 2kbEF1α启动子 ,插入到pCMV mINS的NruⅠ和HindⅢ位点中 ,获得重组质粒pEF1α mINS ;将pCMV mINS和pEF1α mINS分别转染BHK细胞 ,用G418筛选 ,阳性克隆传至 2 0代后 ,分别用放免方法和免疫组化法分析胰岛素和 或胰岛素原在BHK细胞中的表达情况。结果 经放免测定 ,pCMV mINS和pEF1α mINS在BHK细胞中胰岛素和 或胰岛素原的表达量分别为 4 0 77μIU ml和 6 897μIU ml。经免疫组化分析 ,pCMV mINS在BHK细胞质中胰岛素表达水平的灰度值为 190 0± 19 5 6 ;pEF1α mINS在BHK细胞质中胰岛素表达水平的灰度值为 181 4± 18 45 ,在BHK细胞核中表达水平的灰度值为 15 5 4± 11 6 6。结论 在BHK细胞中启动子EF1α启动胰岛素基因表达的活性比启动子CMV高。

Objective To construct eukaryotic higher expression vector of human insulin. Method The recombinant expression plasmid pEF1α|mINS was constructed according to the routine method. pCMV|mINS and pEF1α|mINS were transfected into BHK cells by lipofectin reagent respectively. And the expression products of insulin gene were detected by radioimmunoassay (RIA) and immunohistochemistry when the positive clones were passed to 20th generation. Results The expression levels of insulin and/or proinsulin were 4.077μIU/ml and 6.897μIU/ml respectively detected by RIA. The grey level of insulin expressed from pCMV-mINS was 190.0±19.56 in cytoplasm, from pEF1α-mINS was 181.4±18.45 in cytoplasm, and 155.4±11.66 in nucleus. Conclusion The promoter EF1α was more effective than CMV in BHK cells.